Project Summary (Abstract): The overall goal of this research is to develop the genetic and bacterial systems to investigate the role of the widely encountered bacterial ?bet-hedging? strategy in the adaption to fluctuating environments. In the bet- hedging strategy, the bacterial colony bets that optimal growth conditions may not be sustained. To protect itself against these potential future threats, a very few number of individual cells, which remain isogenic with the rest of the colony, behave as if they sensed a stressful environment even prior to encountering the stress. Such environments could include changes in pH, temperature fluctuations, carbon source deprivation, or antibiotics. Currently there are no techniques to identify, track and study these rare bet-hedging cells in vitro or in vivo. Therefore, in order to achieve our research goal, we intend to design and validate a method to track individual bacteria that transiently express rare phenotypes. Once we are able to study these bet-hedgers before and after the stress is applied, we will be able to determine: i) what fraction of the population express this phenotype, ii) the number of cells expressing this altered phenotype that can be tolerated by the population, iii) what alterations in transcription, protein expression, enzyme pathways and metabolome contribute to this behavior, iv) whether these cells lie on the pathway to mutations that fix adaptive phenotypic traits in the emergent population, and v) the effect that delays in sensing the environment and the mean and variance of stochastic expression have on the cell types that eventually survive the environmental fluctuation. By the end of the two-year R21 grant we are confident that we will have developed a robust, useful cell lineage tracking system for E. coli.